The B cell antigen receptor complex is composed of membrane immunoglobulin noncovalently associated with heterodimers of Ig-α and Ig-β. These signal transducing subunits contain a conserved ITAM motif (immunoreceptor tyrosine-based activation motif) required for signal transduction (Cambier, 1995). Aggregation of the BCR by multivalent antigen initiates transphosphorylation of the Ig-α and Ig-β ITAM motifs and activation of receptor-associated kinases (for review see DeFranco, 1997; Kim et al., 1993; Kurosaki, 1997). Phosphorylated ITAMs recruit additional effectors such as PI3-K, PLC-γ and members of the Ras/MAPK pathway. These signaling events are responsible for B cell proliferation, and increased expression of activation markers such as MHC class II and CD86, that are required to prime the B cell for subsequent interactions with Th cells.
The B cell repertoire is finely tuned to contain maximal receptor diversity in the absence of autoreactivity. Autoreactive clones are eliminated by processes including clonal deletion by apoptosis or receptor editing, and anergy (Goodnow et al., 1988; Hartley et al., 1993; Hertz and Nemazee, 1997; Nemazee and Burki, 1989; Rathmell et al., 1996). In the latter case, autospecific cells persist but are unresponsive to antigen. The molecular mechanisms underlying B cell unresponsiveness have been studied in BCR transgenic mice and in several in vitro models of receptor desensitization (Brunswick et al., 1994; Cambier et al., 1988; Cambier et al., 1990, Erikson et al., 1991; Gay et al., 1993; Nemazee and Burki, 1989; Okamoto et al., 1992; Vilen et al., 1997). Studies in the HEL/anti-HEL double transgenic mouse have shown that B cells tolerant to self antigen exhibit reduced cell surface expression of IgM, are no longer capable of antigen-induced CD86 expression, and are sensitive to Fas mediated apoptosis (Goodnow et al., 1989; Ho et al., 1994; Rathmell et al., 1996). In a number of these models, receptor desensitization is characterized by the inability of antigen to elicit tyrosine phosphorylation or renewed Ca2+ mobilization despite the continued expression of antigen binding receptors.
Recently, disruption of receptor proximal signaling events have been studied in desensitized B cells (Cooke et al., 1994; Vilen et al., 1997). Results from these studies reveal a lack of antigen induced phosphorylation and activation of receptor associated kinases such as Lyn, Blk, and Syk. Johnson et al. showed that receptor-associated kinases could be activated by exposure to doubly phosphorylated ITAM peptides, suggesting that the failure of desensitized receptors to activate signaling pathways was not due to a defect intrinsic to the kinase, but rather reflected a defect at the level of the receptor and its ability to couple to Lyn (Johnson et al., 1995). Johnson et al. hypothesize that receptor unresponsiveness may be due to an uncoupling of Lyn from an otherwise intact mIg/Igαβ complex, or alternatively, a result of excessive phosphotyrosine phosphatase activities at the receptor.
Despite considerable research in this area, previous investigators have failed to teach or suggest the molecular event that the present inventors have shown to be responsible for maintaining the unresponsive phenotype of desensitized receptors. Therefore, prior to the present invention, therapeutic compounds which specifically target this molecular event have not been identified.
A wide variety of medical treatments require regulation of the immune response in a patient. Such treatments include, for example, vaccinations, treatments for autoimmune diseases, immunodeficiency diseases, immunoproliferative diseases, and treatments involving the transplantation of organs and skin. Traditional reagents and methods used to regulate a subject's immune response often results in unwanted side effects. For example, immunosuppressive reagents such as cyclosporin A, azathioprine, and prednisone are used to suppress the immune system of a patient with an autoimmune disease or patients receiving transplants. Such reagents, however, suppress a patient's entire immune response, thereby crippling the ability of the patient to mount an immune response against infectious agents not involved in the original disease. Due to such harmful side effects and the medical importance of immune regulation, reagents and methods to regulate specific parts of the immune system have been the subject of study for many years.
The present invention can be used to overcome traditional problems with immunoregulatory reagents by targeting specific cells and immune receptors in vivo.